Research Article Prostacyclin Synthase: Upregulation ...downloads.hindawi.com/journals/mi/2015/654151.pdf · Prostacyclin (PGI 2) plays a critical role in nephrogenesis and renal

Research ArticleProstacyclin Synthase: Upregulation duringRenal Development and in Glomerular Disease as well as ItsConstitutive Expression in Cultured Human Mesangial Cells

Thomas Klein,1,2 Günther Klaus,1 and Martin Kömhoff1

1Department of Pediatrics, University of Marburg, 35043 Marburg, Germany2Boehringer Ingelheim, Pharma Division Research, 88397 Biberach, Germany

Correspondence should be addressed to Martin Kömhoff; [email protected]

Received 7 August 2014; Revised 2 December 2014; Accepted 3 December 2014

Academic Editor: Aihua Zhang

Copyright © 2015 Thomas Klein et al. This is an open access article distributed under the Creative Commons Attribution License,which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Prostacyclin (PGI2) plays a critical role in nephrogenesis and renal physiology. However, our understanding of how prostacyclin

release in the kidney is regulated remains poorly defined. We studied expression of prostacyclin synthase (PGIS) in developingand adult human kidneys, and also in selected pediatric renal diseases. We also examined PGI

2formation in human mesangial

cells in vitro. We observed abundant expression of PGIS in the nephrogenic cortex in humans and in situ hybridization revealedan identical pattern in mice. In the normal adult kidney, PGIS-immunoreactive protein and mRNA appear to localize to mesangialfields and endothelial and smooth muscle cells of arteries and peritubular capillaries. In kidney biopsies taken from pediatricpatients, enhanced expression of PGIS-immunoreactive protein was noted mainly in endothelial cells of patients with IgA-nephropathy. Cultured human mesangial cells produce primarily PGI

2and prostaglandin E

2, followed by prostaglandin F

2𝛼

Cytokine stimulation increased PGI2formation 24-fold. Under these conditions expression of PGIS mRNA and protein remained

unaltered whereas mRNA for cyclooxygenase-2 was markedly induced. In contrast to its constitutive expression in vitro, renalexpression of prostacyclin-synthase appears to be regulated both during development and in glomerular disease. Further researchis needed to identify the factors involved in regulation of PGIS-expression.

1. Introduction

Prostacyclin synthase (PGIS) is an atypical cytochromep450 enzyme [1], which generates prostacyclin (PGI

2) from

prostaglandin H2(PGH

2), provided by cyclooxygenase-1

(COX1) or COX2. Prostaglandin-I synthase is expressed con-stitutively, consistent with its TATA-less and GC-rich pro-moter [2]. Modulation of constitutive expression with strongupregulation has been observed in uterine development [3].However, in vitro studies have failed to identify specific fac-tors that induce consistently PGIS-protein expression. Pros-tacyclin, which has a half-life of 30 sec in vivo, activates ade-nylate cyclase through interaction with its G-protein-coupledreceptor, dubbed IP [4]. In contrast to certain syntheticand stable prostacyclin analogues, known to activate thenuclear receptor peroxisome proliferator-activated receptor,

𝛽/𝛿 (PPAR-𝛽/𝛿) [5], there remains controversy over whetherthis is also true for endogenous prostacyclin [6, 7].

Development of severe glomerular, vascular, and inter-stitial abnormalities in PGIS-knockout-mice only serves toconfirm the critical role that prostacyclin plays in normalrenal development [8]. Such defects are not observed in theIP-knockout-mice [9], fitting with the notion of a secondPGI2-receptor, possibly PPAR-𝛽/𝛿. Numerous glomerular

actions of PGI2have been reported, including effects on

local hemodynamics, renin secretion, cell proliferation, andmatrix turnover [4].The potential beneficial effects of varioussynthetic ligands on slowing progression of renal disease havebeen observed in experimental animal models as well as inhumans [4].

Despite the fact that prostacyclin has been shown to playseveral roles in the kidney, the pattern of PGIS expression in

Hindawi Publishing CorporationMediators of InflammationVolume 2015, Article ID 654151, 9 pageshttp://dx.doi.org/10.1155/2015/654151

2 Mediators of Inflammation

humans during renal development and in glomerular diseaseis virtually unknown. There are also few studies describingthe in vitro expression of prostacyclin synthase. In humanendothelial cells, biosynthesis of prostacyclin is controlledprimarily through the induction of cyclooxygenase by growthfactors or mitogens whereas expression of PGIS remainsunchanged [10]. Others have shown that bovine endothelialcells translate tumour necrosis factor (TNF) binding into anincrease of COX2 enzyme expression and subsequently intothe induction of prostacyclin synthase mRNA [11]. Finally,incubation of human endothelial cells with acidic fibroblastgrowth factor in the presence of heparin resulted in amarked diminution in PGI

2synthesis caused by a decrease in

expression of both prostacyclin synthase and cyclooxygenaseprotein [12].

Most studies that have reported on prostacyclin synthesisin the scientific literature have focused on expression andregulation of cyclooxygenases, the enzymes that providePGH2and thus cyclooxygenases are considered to be the

rate-limiting step during prostanoid synthesis. Two isoformsof cyclooxygenase have been identified: a constitutive COX1that is thought to be involved in housekeeping functions ofprostaglandins and an inducible COX2 that is believed to beinvolved in the synthesis of high amounts of prostaglandinsunder pathological conditions [13].

The present series of studies were conducted under vari-ous different circumstances in order to gain further insightinto the regulation of prostacyclin synthase. We analyzedthe mRNA and protein expression of prostacyclin synthasein normal “developing” and adult human kidneys, as wellas in glomerular disease. We also investigated prostacyclinsynthesis and synthase in human mesangial cells in vitro.

2. Material and Methods

Thecurrentworkwas conductedwith permission of our insti-tution’s local medical ethics committee. During the courseof normal clinical practice, renal tissue was obtained fromthe unaffected poles of kidneys surgically removed as partof treatment of renal carcinoma (𝑛 = 5 patients). Humanfetal tissuewas collected frommedical abortions [14]. Unusedparaffin-embedded renal biopsy tissue samples remainingafter sectioning were also identified for study. In total 21patients, 12 patients with IgA-nephropathy, seven patientsundergoing routine renal biopsy, one patient with chronicrenal transplant rejection, and one patient with focal seg-mental glomerulosclerosis provided consent for their biopsysamples to undergo the study.

2.1. Immunohistochemistry of Paraffin-Embedded AdultHuman Tissue and Renal Biopsies. Specificity of all primaryantibodies has been rigorously tested and confirmed by colo-calization studies of the respective mRNA using radioactivein situ hybridization [15, 16]. Sections were incubated withrabbit anti-PGIS polyclonal antibodies, as described previ-ously [16]. Primary anti-cyclooxygenase antibodies wereobtained from Santa Cruz (Santa Cruz, CA: COX1, C20:sc#1752, and COX2: C-20, sc#1745).

2.2. Immunohistochemistry of Human Fetal Tissue. The char-acterization of the monoclonal antibodies for prostacyclinsynthase has been published previously [17]. In brief, approx-imately 5 𝜇m tissue sections were sliced from snap-frozensamples, thaw-mounted on poly-L-lysine-coated glass slides,air-dried, and fixed in acetone for 10min at 4∘C.The presenceof primary antibodies was confirmed with the alkaline phos-phatase antialkaline phosphatase method using rabbit anti-mouse or mouse anti-rabbit antibodies.

2.3. Generation of 35S-Labelled Riboprobes and In SituHybrid-ization. Antisense and sense probes for human prostacy-clin mRNA were prepared as follows. A polymerase chainreaction (PCR) fragment (420 base pairs) generated theamplification of mesangial mRNA with the primer pairdepicted below that was ligated and cloned in a pCR 2.1 plas-mid (Invitrogen, USA). The following primers were used togenerate amurine-PGIS riboprobe: forward-GGCTGGCTG-GGTTGAGAATC and reverse-GACCGTGCGAAGGTT-GGTAT. Cloned cDNA fragments were sequenced accordingto the dideoxy method to confirm the identity and orienta-tion of the inserts. In situ hybridization was performed asdescribed previously [16]. After development in Kodak D-19, slideswere counterstainedwith hematoxylin. Photomicro-graphs were taken with a Zeiss Axioskop microscope usingbright field optics.

2.4. Human Mesangial Cells. Normal human kidney tissuewas obtained from tumor nephrectomy surgery. Glomeruliwere obtained from different donors by passage throughserially graded sieves and incubated in growth mediumwhich consisted of RPMI-1640 supplemented with insulin(5 𝜇g/mL), transferrin (5 𝜇g/mL), sodium selenite (5 𝜇g/mL),L-glutamine (1%), penicillin (100U/mL), and streptomycin(100 𝜇g/mL) containing 10% fetal calf serum and HEPES(20mM). Cellular outgrowths of mesangial appearance weresubcultivated and characterized as follows: (a)morphologicalcriteria with elongated appearance; (b) staining with antivi-mentin; (c) staining with smooth muscle cell actin; and (d)absence of staining with anti(factor VIII) and antidesmin.Cells were grown to confluence and growth was arrested bylow serum conditions (0.5% fetal calf serum) in the absenceof supplements for 24–36 h. All experiments were performedusing cells between the third and sixth passages.

2.5. Cell Culture. Cell culturemedia were obtained fromPAA(Coelbe, FRG). Recombinant human (rh) interleukin (IL)types 1𝛽 IΛ-1𝛽 and rh-TNF𝛼were purchased from Endogene(MA, USA). A commercial enhanced chemiluminescencedetection kitwith nitrocellulosemembranes (Hybond-C)wasobtained from Amersham (Braunschweig, FRG). Diclofenacand bovine serum albumin were from Sigma (Deisenhofen,FRG). Primers were synthesized by MWG-Biotech (Ebers-berg, FRG);AmpliTaq polymerasewas obtained fromPerkin-Elmer (Weiterstadt, FRG) and SuperScript reverse transcrip-tase from GIBCO (Eggenstein, FRG). Insulin, transferrinsupplements, and recombinant human interferon (IFN)type-𝛾 rh-IFN-𝛾 were from Boehringer (Mannheim, FRG).

Mediators of Inflammation 3

(a) (b)

(c) (d)

Figure 1: Mesenchymal expression of prostacyclin synthase in the developing human (24 weeks of gestation) and murine (postnatal day 0)kidney. (a) shows a low power view of a developing human kidney. (b) High power reveals absence of PGIS in epithelial structures. (c) Lowpower shows high subcapsular expression of PGIS-mRNA. (d) confirms the absence of labeling over epithelial structures.

Primary antibodies to characterize cellular cultures wereobtained from Dako (CA, USA); secondary antibodies werefrom Dako and Dianova (Hamburg, FRG).

2.6. Western Blot and mRNA Analysis. Analysis of prosta-cyclin synthase expression was performed on human mesan-gial cells. In brief, lysates (80𝜇g) of cells stimulated for20 h with rh-TNF𝛼 (100 ng/mL), IFN-𝛾 (350U/mL), IL-1𝛽(1 nM), or control were solubilized in phosphate bufferedsaline containing 1% Triton X-100. Equal amounts of pro-tein were separated by 10% SDS-PAGE. Immunoblot anal-ysis was performed with different purified polyclonal anti-bodies against PGIS as described previously [17]. Total RNAwas isolated frommesangial cells using the guanidinium thi-ocyanate method with acidic phenol. Reverse transcriptionand PCR were performed as described recently for COX1,COX2, 𝛽-actin, and thromboxane synthase. Accordingly, forprostacyclin synthase, 1 𝜇g of total RNA was used for target-specific reverse transcription with reverse transcriptase andthe primer (5-ATGCGGTAGCGGACGACGGGCACG-3).Polymerase chain reaction amplification was performed

using the cDNA with the antisense (5-ctgcatcagaccgaagccctacctg-3) and sense primer (5-TGCTGAGTGAGAGCC-TCAGGCTTA-3). Reactions were cycled 30 times (30 secat 94∘C, 30 sec at 56∘C, and 30 sec at 72∘C following a 5mindenaturing step at 95∘). Amplification productswere analyzedby 1.5% agarose gel electrophoresis and ethidium bromidestaining. The identity of the fragments was evaluated bytheir molecular mass and dideoxy sequencing. Samples wereassayed at various dilutions to ensure proportionality in theyield of PCR products.

Determination of prostanoids by gas chromatography isas follows. Prostanoids in the supernatant of mesangial cellswere measured by gas chromatography coupled to dual massspectrography (GC/MS/MS) as described previously [18].

3. Results

3.1. Intrarenal Localization of Prostacyclin Synthase

Nephrogenesis. In the developing human kidney, intenseexpression of PGIS was observed in mesenchymal cells ofthe nephrogenic cortex (Figure 1(a)). Epithelial structures


(a) (b)

(c) (d)

Figure 2: PGIS mRNA localizes to mesangial fields ((a), arrow) and endothelial cells of an interlobar artery ((b), arrow). Expression of PGISprotein and mRNA in normal adult kidney. (c) Low power view shows expression of PGIS in endothelial and smooth muscle cells of anarcuate artery (arrow). (d) shows expression of PGIS in cells of the juxtaglomerular apparatus (arrow), adjacent to the macula densa.

corresponding to various stages of renal development andtubular structures were not labeled (Figure 1(b)). An identicalpattern was observed when analyzing prostacyclin synthasemRNA expression in the mouse (Figure 1(c), low powerview). In situ hybridization revealed strong cortical labelingin kidneys on postnatal day 0. Within the exception ofvascular structures, medullary areas showed significantly lesslabeling. High power views confirmed labeling over intersti-tial cells and sparing of epithelial structures (Figure 1(d), highpower view).

Normal Adult Kidney. Radioactive in situ hybridizationrevealed specific signals for prostacyclin synthase in themesangial region of glomeruli (Figure 2(a)) and over arterialendothelial cells (Figure 2(b)). As expected from biologicaland pharmacological studies, PGIS immunoreactivity wasdetected in endothelial cells of blood vessels (Figure 2(c)) ofnormal kidney as well as in the smooth muscle cells of arter-ies. Staining of PGIS in the glomerulus was comparativelylow in normal human tissue. Note PGIS immunoreactivity incells of the juxtaglomerular apparatus adjacent to cells of themacula densa (Figure 2(d)).

Pediatric Renal Disease. Glomerular expression of PGIS-immunoreactive protein varied considerably in tissues fromchildrenwith renal disease ranging from completely absent tostrong and localized primarily to endothelial cells. However,there was no evidence that would link expression with aparticular disease. Shown are high power views fromapatient

with IgA-nephropathy (Figure 3(a)), minimal change diseasewith focal segmental glomerulosclerosis (Figure 3(b)), andchronic transplant rejection (Figure 3(b)). Strong capillaryexpression of prostacyclin synthase in renal medulla wasobserved in the same biopsy (Figure 3(d)).

3.2. Prostacyclin Synthesis in Cultured HumanMesangial Cells

In Vitro Studies. The profile of prostanoid synthesis inour cultured human mesangial cells (HMCs) is shown inFigure 4. Under basal conditions, mesangial cells producedpredominantly PGI

2determined as 6-keto-PGF

1𝛼, the pri-

mary product of PGI2metabolic breakdown. Considerably

smaller amounts of PGE2and PGF

2𝛼 were observed and

thromboxane (TX) type B2, the stable product of Tx type

A2, was only barely detectable. Incubation of growth-arrested

HMC from different donors with cytokines consisting ofIL-1𝛽 (1 nM), TNF𝛼 (10 ng/mL), and IFN-𝛾 (250U/mL)resulted in increased expression of prostacyclin (24-fold) andPGE2(65-fold). There was no apparent change in TxB

2and

PGF2𝛼 production. Coincubation of stimulated HMC with

the nonsteroidal anti-inflammatory drug, diclofenac (1𝜇M),resulted in a complete inhibition of prostanoid synthesis.In contrast, addition of the glucocorticoid dexamethasone(1 𝜇M) lowered cytokine-stimulated prostaglandin produc-tion to the same levels as seen under control conditions. Theprofile of exogenously added arachidonic acid metabolismis shown in Table 1. In a concentration-dependent manner,


(a) (b)

(c) (d)

Figure 3: Expression of prostacyclin synthase in various renal diseases. (a) IgA-nephropathy, (b) focal segmental glomerulosclerosis.Glomerular (c) and medullary (d) expression of PGIS in endothelial cells and peritubular capillaries in a transplanted kidney with chronicrejection.

###

ns ns

###ns ns

# ns

6-KetoPGE2

TxB2

0

500

20000

10000

1000

30000

CytokinesControl Cyto. +diclo. Cyto. +dex.

Pros

tano

ids (

pg/106

cells

)

∗∗∗

∗∗∗

######

PGF2𝛼PGF1𝛼

Figure 4: Pattern of COX1- and COX2-dependent prostanoidformation of human mesangial cells. HMCs were incubated for20 h with cytokines in the absence or presence of diclofenac(1 𝜇M), dexamethasone (1 𝜇M), or vehicle. Prostanoid formationwasmeasured in the supernatant. Data represent means (pg/106 cells) ±SEM (𝑛 = 5). One-way analysis of variance Bonferroni’s multiplecomparison test was performed for each product. ∗∗∗𝑃 < 0.001versus control; ###𝑃 < 0.001 and #𝑃 < 0.05 versus cytokines; ns:nonsignificant.

conversion to prostacyclin and PGE2occurs. Once again,

the levels of TxB2and PGF

2𝛼, prostanoids known to exert

vasoconstrictive actions, were hardly detectable, even whenthe HMCs were challenged with arachidonic acid (20 𝜇M).

To gain further insight into the regulation of prosta-cyclin synthase activity following cytokine stimulation, thecyclooxygenase step was bypassed by the addition of exoge-nous PGH

2, the immediate substrate for PGIS. As can be

seen from the data in Table 2, compared to control, therewas no significant difference in the conversion of PGH

2to 6-

keto-PGF1𝛼 in any of the cytokine or glucocorticoid treated

samples.These data point towards a constitutive activity of prosta-

cyclin synthase. To investigate this aspect at the protein level,Western blot analysis was performed using cell lysates ofcytokine treated HMC or controls. The authenticity of theobserved single band of approximately 52 kD was evidencedusing the purified bovine enzyme as a positive control(Figure 5(a)).

Similar results, confirming the constitutive nature of theenzyme, were obtained addressing the corresponding mRNAexpression (Figure 5(b)).Messenger RNAexpression for bothPGIS and COX1 was unaffected by cytokine stimulation,whereas expression of COX2 mRNA was markedly induced.


Table 1: Prostanoid formation of cytokine-stimulated HMC from different concentrations of exogenously added arachidonic acid.

6-Keto-PGF1𝛼 PGE

2TxB2

PGF2𝛼

AA (0 𝜇M) 242 ± 42∗∗∗ 78 ± 8∗∗∗ 96 ± 7ns 40 ± 13ns

AA (2 𝜇M) 6590 ± 300∗∗∗ 6120 ± 370∗∗∗ 110 ± 7ns 560 ± 6.6ns

AA (5 𝜇M) 11350 ± 250∗∗∗ 10390 ± 610∗∗∗ 83 ± 7ns 116 ± 7ns

AA (20 𝜇M) 14800 ± 100∗∗∗ 13500 ± 200∗∗(a) 100 ± 24ns 226 ± 40∗∗(b);##(c)§(d)

HMC were stimulated with cytokines for 20 h and later they were incubated with the depicted concentrations of exogenous arachidonic acid (AA) or onlybuffer (0 𝜇M) for 15min at 20∘C. Prostaglandins generated were extracted from the supernatant as described and analyzed by GC/MS/MS method. Valuesare depicted as means ± SEM (pg/106 cells) of three experiments. One-way analysis of variance Bonferroni’s multiple comparison test. ∗∗∗𝑃 < 0.001 for allcombinations. (a)∗∗𝑃 < 0.01 for 5 𝜇Mversus 20 𝜇M; (b)∗∗𝑃 < 0.01 for 0 𝜇Mversus 20 𝜇M; (c)##𝑃 < 0.01 for 2 𝜇Mversus 20𝜇M, and (d)§𝑃 < 0.05 for 5 𝜇Mversus20𝜇M; ns: not significant.

Table 2: Conversion of the precursor endoperoxide PGH2to 6-

Keto-PGF1𝛼 in the presence of cytokines or dexamethasone.

6-Keto-PGF1𝛼 (ng/106 cells)

Control 197 ± 14Cytokines 139 ± 10∗

Cytokines + dexamethasone (1mM) 144 ± 7∗

HMC were stimulated for 20 h with cytokines, cytokines plus dexametha-sone, or vehicle. Following stimulation, the medium was aspirated and thecells were stimulated for 5min at 20∘C with 20𝜇MPGH2 in phosphatebuffered saline. The spontaneous decay of PGH2 in aqueous solution from acontrol experiment was considered. The figure consists of the mean of threeexperiments ± SEM. One-way analysis of variance Bonferroni’s multiplecomparison test: ∗𝑃 < 0.05 versus control.

Interestingly, RT-PCR analysis failed to detect mRNA forTXS and suggested a negligible role for TxA

2as mesangial

cell derived prostanoid. The extensive increase of induciblenitric oxide synthase mRNA under identical conditions wasreported in HMC [19] and served as a control for cytokineactivity.

4. Discussion

In the present study, it appear to be a strong expression ofPGIS mRNA and immunoreactive protein in mesenchymalcells of developing human and mouse kidney, respectively.The greatest expression appeared to be subcortical, in themost immature part of the kidneys, namely, the nephrogeniccortex, in mesenchymal cells adjacent to ureteric buds. Previ-ously, the regulation of PGIS expression during developmenthas been shown during uterine development, with high levelsof expression occurring in the most immature cells [3].The strong expression of PGIS suggests that promoters ofPGIS gene expression that have yet to be identified must beinvolved. Further studies, possibly including the investigationof miRNAs as potential regulators, are needed to identify themechanisms leading to the upregulation of PGIS expression.Considering the multiple defects in renal development inPGIS-ko mice, including increased interstitial fibrosis [3],leads to speculation that prostacyclin plays an important rolein the prevention of fibrosis and that this process may bemodulated via PPAR-𝛽/𝛿 (given the normal renal phenotypein IP-ko-mice [9]).

Our study describes for the first time the intrarenallocalization of prostacyclin synthase mRNA and immunore-active protein in the healthy adult human kidney. Expressionof PGIS mRNA and immunoreactive protein in vascularstructures appears to confirm pharmacologic findings and isconsistent with a role for prostacyclin as a vasodilator andinhibitor of platelet activation [20]. Expression of PGIS inthe juxtaglomerular apparatus is in accordance with prosta-cyclin as mediator of renin release [21]. Expression of PGISmRNA and immunoreactive protein was less prominentand discernible over mesangial fields. The exact cellularlocalization (mesangial or endothelial cells) could not beidentified. Consistent with previous reports [22], there wasno observable tubular expression of PGIS. This agrees withthe findings of earlier studies which did not show a role ofendogenous prostacyclin on tubular transport [20].

We investigated whether PGIS, like other developmen-tally regulated genes [23], is upregulated in glomerulardisease. We observed variable degrees of PGIS expression inhuman kidney disease, though the expression was frequentlyhigher than that seen in samples from healthy tissue controls.The interpretation of these observations was limited by thenumber of biopsies available for study and the multipleunderlying diseases, precluding any correlation of PGISexpression with a particular glomerulopathy.The majority ofcases showed upregulation of PGIS in glomerular and per-itubular endothelial cells, which suggests a common inducer(Figures 3(a) and 3(b)). In some biopsies, however, inter-(Figure 3(c)) and intraglomerular (Figure 3(d)) expressionwas quite variable, possibly altered by local hemodynamics.Serial sections stained for COX1 and COX2 (data not shown)did not reveal coexpression with PGIS, arguing for differen-tial modes of regulation (of COX1, COX2, and PGIS, resp.).As endothelial cells, for instance, are known to synthesizeprostacyclin, absence of COX1 orCOX2 costainingwith PGISmay simply reflect high specificity and reduced sensitivity ofour immunostaining procedure.

Our observations with cultured human mesangial cellsappear to confirm that prostacyclin [24] is the predominantmesangial prostanoid both under basal conditions and fol-lowing cytokine stimulation. The increase in PGI

2synthesis

was mirrored by PGE2synthesis [24]. Similarly to PGI

2,

PGE2is a potent vasodilator, dependent on specific receptors

located on target cells.


94

67

43

(a)

COX1

Cyt Cyt Cyt Cyt Cyt CytCC C C C C C

500400300200100

COX2 TXS PGIS iNOS 𝛽-Act

(b)

Figure 5: (a) Western blotting of prostacyclin synthase in HMC. Cell lysates (80𝜇g) of cytokine treated HMC or control were stained witha polyclonal antibody for PGIS. Lane 1: control; lane 2: cytokine stimulation; lane 3: positive control (partially purified protein of bovinePGIS with a molecular mass of 52 KD); lane 4: molecular marker. (b) mRNA analysis of prostanoid generating enzymes and iNOS in HMC.RT-PCR analysis of the mRNA for COX1, COX2, TXS, PGIS, and iNOS from quiescent human mesangial cells (2 × 106) was treated for 20 hwith cytokines or vehicle. RNA preparation and PCR analysis were performed as described inMaterial andMethods. A 100 bp ladder is givenon the right to evaluate the mass of the amplified fragments.

Despite using highly selective and sensitive GC/MS/MS,we could only detect trace amounts of the vasoconstrictingprostanoids, TxA

2and PGF

2𝛼, from HMS. In contrast to

cytokine induced PGE2and PGI

2formation, we did not

observe changes in TxA2and PGF

2synthesis. There is evi-

dence that these prostanoids are predominately formed pro-ducts of human glomerular epithelial cells (unpublishedobservations). Thus, we suggest that the previously reported[25] formation of TxA

2or PGF

2𝛼 from human mesangial

cells is possibly a consequence of contamination of theprevious authors’ work with visceral epithelial cells. The pre-dominance of the vasodilatory prostanoids PGE

2and PGI

2

suggests that mesangial prostanoid formation exerts a tonicvasodilatory effect on the glomerular capillary network.

In a different set of experiments, we sought to betterunderstand the mechanisms involved in cytokine inducedPGI2synthesis. Exposure of HMC to cytokines resulted

in a 24-fold increase of PGI2synthesis, but there was no

evidence of PGIS mRNA and protein having been induced.In contrast, there was a marked induction of COX2 mRNAand protein. These results are in agreement with previousstudies [26] and suggest that the limiting step in HMC-derived PGI

2synthesis is the regulation of COX2 expression.

To substantiate the concept that COX2 levels regulate pros-tanoid synthesis in HMCs, we investigated the effect of dex-amethasone on cytokine induced PGI

2prostaglandin forma-

tion. Dexamethasone is known to inhibit stimulated expres-sion of both COX2 protein and mRNA through its action ontranscriptional and posttranslational mechanisms. In cytok-ine-stimulated HMC, dexamethasone repressed COX2mRNA and protein expression in parallel with a decrease inprostanoid formation to levels seen inside control. As dex-amethasone failed to affect expression of the PGIS gene, ourdata appears to additionally support the concept that COX2is the key step in controlling prostanoid formation in HMCs.Exposure of cytokine-stimulated HMCs to the nonsteroidalanti-inflammatory drug diclofenac, which inhibits both

COX1 and COX2 activities, reduced prostanoid synthesis tolevels below those seen in controls. These findings appear tosuggest that COX1 contributes to basal HMC-derived pros-tanoid formation. Accordingly, we also observed constitutiveexpression of COX1 mRNA.

Our data appear to be in contrast with the observationsmade in bovine aortic endothelial cells, which has demon-strated the upregulation of prostacyclin mRNA followingTNF𝛼 stimulation [11]. However, our findings are in linewith those from experiments conducted in human venousumbilical endothelial cells [10]. Therefore, we propose thatcell-specific regulation of prostacyclin synthase may occurunder certain conditions or that the apparent difference infinding is a consequence of species differences. This notionis a supported pattern of organization of the human prosta-cyclin synthase gene, which was recently determined anduncovered consensus sequences for Sp-1, Ap-2, and, interest-ingly, NF-𝜅B in the promoter region [11]. NF-𝜅B is knownas a transcriptional activator involved in the transmission ofproinflammatory responses and could be a target for PGIStranscriptional regulation. The signalling pathway triggeringPGIS induction in human systems therefore seems to bemorecomplex and may involve a complex cytokine network (ina fashion similar to that recently proposed for nitric oxidesynthase) or the presence of yet unknown mediators.

Nevertheless, our data appear to demonstrate clearly thatregulation of PGI synthesis is regulated predominantly byCOX2 activity, which serves to provide PGH to the consti-tutively expressed PGIS.

When considering our current findings in conjunctionwith what is already known about prostacyclin synthase, itappears that our data shows it to be under the influence ofa developmentally regulated gene which is often reexpressedin glomerular disease. These in vivo findings appear to be incontrast with the constitutive expression of PGIS in primaryhuman mesangial cells. Nevertheless, our data point to hith-erto unknown regulators of prostacyclin synthase expression.


Abbreviations

PGIS: Prostacyclin synthaseTXS: Thromboxane synthaseCOX: CyclooxygenaseHMC: Human mesangial cellGC/MS/MS: Gas chromatography/mass

spectrometryPPAR-𝛽/𝛿: Peroxisome proliferator-activated

receptor 𝛽/𝛿IgA-GN: IgA-nephropathyFSGS: Focal segmental glomerulosclerosisPGI2: Prostaglandin I

2

PGH2: Prostaglandin H

2

TNT𝑥: Tumour necrosis factor type alpha

PCR: Polymerase chain reactionIL: InterleukinRh: Recombinant human.

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper.

Acknowledgments

This study was supported by Stiftung P. E. Kempkes, Mar-burg (19-2000 and 26-2001), and Deutsche Forschungsge-meinschaft (KO 1855/2-1) awarded to Martin Kömhoff. Theauthors would like to thank Dr. M. D. Breyer and Dr. L. S.Davis (Vanderbilt University, USA) for the expert advice onhow to perform in situ hybridization.

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Research Article Prostacyclin Synthase: Upregulation ...downloads.hindawi.com/journals/mi/2015/654151.pdf · Prostacyclin (PGI 2) plays a critical role in nephrogenesis and renal

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